Vr 885

Since the prevalence of urogenital C. trachomatis is caused by serovars D to F, we performed our experiments using C. trachomatis serovar D (ATCC VR885) in this study. HeLa cells (ATCC CCL-2) and primary fibroblasts isolated from C57BL/6J mice (kindly provided from Saleh M. Ibrahim, Lübeck Institute of Experimental Dermatology, University of Lübeck) were used as host cells.

C. trachomatis serovar D UW3 (ATCC^© VR-885^™, US) was propagated in McCoy cells as previously described [24 (link)]. Briefly, confluent cell monolayers grown in 25 cm² flasks (6x10⁶ cells/well), were infected with chlamydial EBs by centrifugation at 754 x g for 30 min and harvested by scraping after 36 hours of incubation. The suspension containing Chlamydial EBs was, then, added to equal volumes of 4X Sucrose Phosphate (4SP) buffer and stored at −80°C.
For C. trachomatis titration, confluent cell monolayers, grown on coverslips in 24-well plates (1x10⁵ cells/well), were infected with 10-fold serial dilutions of C. trachomatis EB suspension by centrifugation at 754xg for 30 min, then washed 3x with PBS and added with complete medium. After 36-hours of incubation at 37°C and 5% CO₂, infected cell monolayers were washed 3x with PBS and, then, fixed in 96% ice cold methanol for 10 min at -20°C. Chlamydial inclusions were stained by using DFA via a fluorescein isothiocyanate (FITC)-conjugated anti-Chlamydia lipopolysaccharide (LPS) antibody kit (Oxoid, US), following the manufacturer’s instructions. Inclusions were visualized and counted by using a Leica DM5000B fluorescence microscope (Leica, US) at 400× magnification.

C. trachomatis SvD (Trachoma type D strain UW-3/Cx, ATCC® VR-885™) was propagated in HeLa cells as previously described [27 (link)]. Briefly, the HeLa cells were cultured in six well plates and infected with 1.5 C. trachomatis SvD inclusion forming units (IFU) per HeLa cell. The infection was followed by centrifugation at 750 g for 1 h, and thereafter incubated at 35 °C for 2 h, after which the media was enriched with 0.5 % glucose and 1 μg/ml cyclohexamide. The plates were incubated at 37 °C for 48 h, and thereafter harvested and purified as described by Olsen et al. [27 (link)].
The concentration of infectious bacteria was determined by culturing bacterial suspensions on McCoy-cells [28 (link)]. The plates were incubated at 37 °C for 22 h. Visualization of inclusions was made by incubating the cells with polyclonal rabbit antibodies against chlamydial Major Outer Membrane Protein and chlamydial Heat Shock Protein-60 [29 (link)], and thereafter stained with 4 μg/ml Alexa Fluor 488 labelled goat-anti rabbit antibody (Life Technologies) and kept in the dark at 4 °C until microscopy was carried out.
The cell plates were evaluated using a fluorescent microscope (Olympus IX71). Inclusion bodies were enumerated by counting positively stained inclusions in at least 20 fields in each well using the 40x objective on microscope. Results were calculated as average of duplicate samples.